The long-term goal of the proposed research is to obtain a detailed description of ligand-ribosome interactions during peptide bond formation, based on the hypothesis that the ribosome is a dynamic entity whose moving parts promote translation. Although recent high-resolution cryo-EM and crystallographic images provide new insights into the mechanism of translation, these 'snapshots' do not explain how the ribosome interacts with its numerous ligands or how structural changes in the ribosome facilitate this process. The specific objectives of this proposal are to define the structural and functional relationships between tRNA and the components of the peptidyl transferase center (PTC) and the exit site (E site) of the E. coil 50S ribosomal subunit, using chemical, biochemical and genetic approaches. (1) Putative differences in the structural organization of the PTC of archaeal and (eu)bacterial ribosomes, as well as possible changes in the position of tRNA relative to 23S rRNA during peptidyl transfer, will be examined by qualitative and quantitative analysis of the pattern of short-range, photochemically-induced crosslinks to the PTC from the 3' nucleotide of tRNA at the P and A sites and a transition-state analog that spans both. (2) Crystallography and tRNA crosslinking have revealed that there is a protein, L27, at or near the PTC of the (eu)bacterial 50S ribosomal subunit, a region that is otherwise composed entirely of RNA. Moreover, deletion of L27 leads to severe defects in translation. The functional role of L27 will be assessed by investigating the effects of specific truncations on cell growth and ribosome activity, and the site of tRNA crosslinking in L27 will be determined. (3) Protein L1 and its associated RNA comprise a flexible and semi-autonomous domain within the 50S subunit that appears to promote the release of deacyated tRNA from the ribosome following peptide bond formation. To test this theory, the ability of tRNA to interact with L1- RNA fragement complexes will be checked in vitro and the the effects of specific mutations in L1 and the 23S rRNA on tRNA release will be investigated in vivo. (4) As the main contribution of 23S rRNA to the catalysis of peptide bond formation may be to correctly position the peptidyl- and amnioacyl-tRNA substrates, mutations in the 23S rRNA at the PTC may disrupt this process. This hypothesis will be tested by evaluating the pattern of crosslinks between the 3' ends of P- and A-site tRNA and ribosomes containing mutant 23S rRNA. As many antibiotics target the ribosome, a detailed understanding of protein synthesis will contribute to the rational development of new antibiotics which are urgently needed in the face of widespread antibiotic resistance among pathogenic microorganisms.